Tuberculosis is the leading single cause of infectious disease mortality worldwide. Immunity to tuberculosis depends upon the cellular immune system. Hence, developing an improved vaccine will require characterization of T cell antigens recognized early during the course of infection, and an understanding of the mechanisms by which T cells recognize and respond to Mtb infected cells. Two novel proteins (TbH9 and 38.1) from Mtb that are potent T cells antigens have recently been cloned and expressed in our laboratory. We have found that these proteins elicit vigorous T cell proliferation and interferon-gamma production from healthy PPD positive individuals, and that one of these proteins is preferentially expressed in cells infected with live Mtb. However, little is known about the response to these proteins in persons who have been recently infected or in those with active tuberculosis. Additionally, several lines of evidence, including the susceptibility of mice rendered deficient in beta2 microglobulin to infection with Mtb, have suggested an important role for CD8+ cytotoxic T lymphocytes (CTL) in immunity to tuberculosis. Using peripheral blood derived dendritic cells infected with Mtb, we have isolated human CD8+ CTL clones to Mtb. This is the first demonstration of human Mtb specific CD8+ T lymphocytes, and further characterization of these cells will be an important part of this proposal. This proposal rests upon two major hypotheses: 1) Protective T cell responses to Mtb arise early in the course of infection and are associated with the production of interferon-gamma (IFN-gamma), and 2) MHC class Ia restricted and non restricted CD8+ CTL that recognize Mtb infected cells are generated during the course of infection. The specific aims are: 1) To characterize human Mtb specific T cell responses in diseased and healthy infected individuals. 2) To characterize human CD8+ T cell responses to Mtb. The research methods for achieving these goals include a collaborative project with Seattle-King County Tuberculosis Clinic to study human T cell responses to recombinant Mtb antigens during the evolution of disease. In addition, the relative contribution of MHC class Ia restricted and non-restricted CD8+ CTL will be evaluated in healthy PPD positive individuals. The mechanisms by which Mtb gain entry into the MHC class Ia will be explored using selective inhibition of antigen processing. The mechanisms of antigen processing and nature of the antigen or MHC class Ia non-restricted clones will also be evaluated. Finally, mammalian expression vectors for the proteins TbH9 and Ag 85b will be constructed, and used to elicit antigen specific CD8+ CTL. The proposed studies will allow for greater understanding of T cell responses to tuberculosis.